Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

ABSTRACT

This invention relates to novel adenoviruses useful in the production of high titers of recombinant adeno-associated virus (rAAV) comprising a foreign DNA insert and methods of making these adenoviruses. The adenovirus comprises the AAV rep gene in which the p5 promoter is replaced by a minimal promoter or by no promoter. The invention also provides methods of producing high levels of rAAV as a substantially homogenous preparation and composition of rAAV.

TECHNICAL FIELD OF THE INVENTION

This invention relates to novel adenoviruses useful in the production ofhigh titers of recombinant adeno-associated virus (rAAV) comprising aforeign DNA insert and methods of making these adenoviruses. Theadenovirus comprises the AAV rep gene in which the p5 promoter of rep isreplaced by a minimal promoter or by no promoter. The invention alsoprovides methods of producing high levels of rAAV as a substantiallyhomogeneous preparation and compositions of rAAV.

BACKGROUND OF THE INVENTION

A recombinant virus carrying a foreign DNA insert may be used to delivergenes to cells, where the gene may be expressed, if desired, to permitproduction of recombinant proteins in vitro or in vivo, vaccination ofhuman and non-human mammals, or treatment or amelioration of diseases orgenetic defects in humans or in non-human mammals. One may treat orameliorate diseases or genetic defects by providing some effective levelof normal gene products, increased levels of gene products or byblocking endogenous production of a gene, whose expression would bedeleterious to the cell or organism.

Methods for delivering an exogenous gene to a mammalian cell include theuse of mammalian viral vectors, such as those that are derived fromretroviruses, adenoviruses, herpes viruses, vaccinia viruses, polioviruses, adeno-associated viruses, hybrid viruses (e.g., hybridadenovirus-AAV, see U.S. Pat. No. 5,856,152) and the like. Other methodsinclude direct injection of DNA, biolistic administration of DNA,electroporation, calcium phosphate precipitation, as well as methods ofadministration which utilize ligand-DNA conjugates, liposome conjugatesof DNA, polycation-DNA complexes or adenovirus-ligand-DNA conjugates.

Adeno-associated virus (AAV) systems have many advantages that can beexploited for delivery of transgenes. AAV is a helper-dependent DNAparvovirus which belongs to the genus Dependovirus. AAV requires helperfunction in order for a productive infection to occur. Helper functionsmay be provided by a number of agents, but generally co-infection withan unrelated helper virus, either adenovirus, herpesvirus or vaccinia,is used. In the absence of such co-infection, AAV establishes a latentstate by insertion of its genome into a host cell chromosome. Subsequentinfection by a helper virus rescues the integrated copy which can thenreplicate to produce infectious viral progeny. AAV has a wide host rangeand is able to replicate in cells from any species so long as there isalso a successful co-infection of such cells with a suitable helpervirus. AAV has not been associated with any human or animal disease anddoes not appear to alter the biological properties of the host cell uponintegration. For a review of AAV, see, e.g., Berns and Bohenzky (1987)Advances in Virus Research (Academic Press, Inc.) 32:243–307.

AAV has a genome of about 4.7 kb in length, including inverted terminalrepeats (ITRs) that often, but not necessarily are 145 nucleotides inlength. The AAV genome encodes two genes, rep and cap, each of whichexpresses a family of related proteins from separate open reading framesand which are produced by alternative mRNA splicing and differenttranscriptional and translational start sites. Rep polypeptides (Rep78,Rep68, Rep52, and Rep40) are involved in replication, rescue andintegration of the AAV genome. Rep78 and Rep68 have the sameamino-terminal sequence and share the same promoter, p5, but Rep78contains an exon that is alternatively spliced out in rep68. Similarly,Rep52 and Rep40 have the same amino-terminal sequence and share the p19promoter, which is downstream from the p5 promoter, but rep52 containsan exon that is alternatively spliced out in rep68. Cap proteins (VP1,VP2, and VP3) form the virion capsid. Cap gene transcription is drivenby the p40 promoter. See FIG. 2B for a schematic diagram of the rep andcap genes and promoters p5, p19 and p40. Flanking the rep and cap openreading frames at the 5′ and 3′ ends of the AAV genome are the ITRs. Incertain AAV genomes, the ITRs are 145 nucleotides in length, the first125 bp of which are capable of forming Y- or T-shaped duplex structures.The entire nucleic acid encoding rep and cap can be excised and replacedwith a transgene [B. J. Carter, in “Handbook of Parvoviruses”, ed., P.Tijsser, CRC Press, pp. 155–168 (1990)]. The ITRs represent the minimalsequence required for replication, rescue, packaging, and integration ofthe AAV genome if other sources of rep and cap are provided.

When AAV infects a human cell, the viral genome integrates intochromosome 19 resulting in latent infection of the cell. Uponintroduction of helper functions into the cell, such as by infectionwith a helper virus, the AAV provirus is rescued and amplified. Therescued AAV genomes are packaged into preformed protein capsids(icosahedral symmetry approximately 20 nm in diameter) and released asinfectious virions that have packaged either + or − single stranded DNAgenomes following cell lysis.

Replacing the rep and cap sequences with a desired transgene yields arAAV capable of delivering the transgene to target host cells. Incurrent methods, the deleted rep and cap sequences are supplied to thehost cells by other viruses or plasmids where they are transiently orstably expressed. There are also a number of cell lines that stablyexpress rep and cap. The host cells also require helper functions inorder for the rAAV to replicate and excise from the host cell genome.The helper functions usually are provided by helper viruses (eitherwildtype or crippled viruses), plasmids containing the helper virusfunctions or physical methods.

Although it is known that rep is required for replication and excisionof AAV, the amount of Rep proteins required for effective rAAVproduction is, as yet, unclear. U.S. Pat. No. 5,354,678 states that Repproteins may be toxic to certain cell lines, and WO 97/06272, WO98/46728 and Li et al. suggest that attenuation of Rep78/68 productionresults in higher levels of production of rAAV. In contrast, other art,such as U.S. Pat. No. 5,658,776, explicitly states that high expressionof Rep proteins—a result of replacing the native p5 promoter with astrong promoter, such as the human immunodeficiency virus long terminalrepeat (HIV LTR)—results in high level expression of rAAV. Similarly,U.S. Pat. No. 5,837,484 states that the p5 promoter should be replacedby a strong constitutive promoter or inducible promoter, such as themetallothionein promoter, in order to overcome the strong feedbackinhibition by Rep of its own transcription. Thus, U.S. Pat. Nos.5,658,776 and 5,837,484 suggest that high expression of Rep78/68 isrequired for efficient rAAV production.

One method that has been used to produce recombinant AAV (rAAV) vectorscomprises co-transfecting eukaryotic cells with a plasmid containingrAAV sequences (the cis plasmid) and a plasmid containing rep and cap(the trans plasmid), and infecting the cells with a helper virus (e.g.,adenovirus or herpes virus). See U.S. Pat. No. 5,753,500. Li et al. (J.Virol. 71:5236–5243, 1997) have modified this method by altering thetranslation initiation codon of the Rep78/68 proteins in the transplasmid to decrease the translation of the Rep protein and increaseproduction of rAAV. However, the disadvantage of the methods taught byU.S. Pat. No. 5,753,500 and Li et al. is that co-transfection of twoplasmids along with infection by a helper virus is inefficient, mayexhibit poor reproducibility, may result in generation ofpseudo-wildtype replication-competent AAV (rcAAV), and cannot be easilyscaled up for industrial production of rAAV. rcAAV, comprising rep andcap flanked by ITRs, is produced when the rep and cap genes recombinewith the ITRs flanking the transgene which results in deletion of thetransgene.

A second method that has been used to produce rAAV involvesco-transfection of three plasmids into eukaryotic cells. In this method,one plasmid carries the transgene and ITRs (the cis plasmid), a secondplasmid encodes the rep and cap genes (the trans plasmid), and the thirdplasmid encodes the helper virus functions, i.e. adenoviral genes suchas E1a, E1b, E2a and E4 (the helper plasmid). The disadvantages of thefirst method are shared with this method.

A third method involves the use of a packaging cell line such as oneincluding AAV functions rep and cap. See U.S. Pat. Nos. 5,658,785 and5,837,484 and PCT US98/19463. The packaging cell line may be transfectedwith a cis plasmid comprising the transgene and ITRs, and infected bywild-type adenovirus (Ad) helper. See U.S. Pat. No. 5,658,785.Alternatively, the packaging cell line may be co-infected by a hybridAd/AAV, in which a hybrid Ad vector carries the cis plasmid in the E1locus (see U.S. Pat. No. 5,856,152), and by a wild-type or mutant Adthat supplies E1. See, e.g., Reference 7. The disadvantage of thismethod is that it requires making a cell line that expresses sufficientlevels of rep and cap, and requires multiple components—including thecell line, the rAAV genome, and an adenovirus—to produce rAAV, which donot lend themselves to easy and convenient downstream manufacturingprocesses. In addition, some of these packaging cell lines do notproduce high levels of rAAV.

A fourth method is provided by a prophetic example in U.S. Pat. No.5,354,678. The method involves using a recombinant adenovirus in whichthe rep and cap genes of AAV replace a part of the adenovirus genome notessential for helper virus functions. In this method, an AAV/EBV plasmidvector comprising an rAAV genome is introduced into a cell to produce anrAAV producer cell. It is presumed that the rep gene is driven by itsnative p5 promoter or by a strong inducible promoter. The recombinantadenovirus comprising rep and cap is then introduced into the cell andtheir production is induced such that rAAV is produced by the cells.U.S. Pat. No. 5,354,678 does not disclose the levels of rAAV, if any,produced by this method.

As described above, current rAAV production methods are not amenable forproduction of sufficient rAAV for pharmaceutical applications in aconvenient manner. However, the problem of reproducibly generating highlevels of substantially homogeneous replication-deficient rAAV by anefficient method that is applicable to large-scale industrial productionis solved by the present invention.

SUMMARY OF THE INVENTION

The instant invention provides an alternative production method thatresults in high yields of rAAV vector and is amenable to large-scaleindustrial applications. The invention provides a novel adenovirusvector comprising rep and cap genes, thus providing AAV rep and cap andadenovirus helper functions in one component. In the adenovirus vectorof the instant invention, the native AAV p5 promoter upstream of rep isremoved and replaced with a minimal promoter or with no promoter. Thisnovel vector, when infected into cells containing a nucleic acidsequence comprising a transgene flanked by AAV ITRs, results in theproduction of high levels of rAAV. The nucleic acid sequence comprisingthe transgene flanked by AAV ITRs may be established in the host cell bystable integration into the host cell chromosome, secondary infectionwith an adenovirus or other viral vector carrying the transgene flankedby ITRs (see, e.g, U.S. Pat. No. 5,856,152), infection with an rAAVcomprising the transgene, or any other method known in the art, such astransfection, lipofection or microinjection, of plasmid DNA comprisingthe transgene flanked by ITRs.

In one embodiment of the invention, rep, operably linked to a minimalpromoter or to no promoter, is inserted into either the E1 or E3 regionsof an adenovirus The adenovirus is deleted in E1 or E3 alone, or acombination of both. In another embodiment, the adenovirus vector isfurther deleted in E4. In this embodiment, rep sequences may be insertedin E4, while upstream of these rep sequences there may be no promoter ora minimal promoter. In a preferred embodiment, cap is inserted alongwith the rep gene into the adenoviral vector. In another aspect of theinvention, the adenoviral vector comprising the minimal promoter orpromoterless rep is used in a method to produce rAAV. The advantage ofthis method is that it is easily scaled for industrial production ofrAAV.

In the method of the invention, the host cell is supplied with an rAAVgenome, and the adenovirus comprising the minimal promoter orpromoterless rep is infected into the cell. In one embodiment of theinvention, the host cell is either simultaneously or sequentiallyco-infected with two adenoviruses, wherein one adenovirus comprises capand rep driven from a minimal promoter or no promoter, as describedabove, and the other adenovirus comprises an rAAV genome. In anotherpreferred embodiment, an adenovirus comprising cap and rep driven from aminimal promoter or no promoter is used to infect a host cell comprisinga stably expressed rAAV genome.

In a preferred embodiment of the invention, the method is one in which ahigh titer of substantially homogeneous rAAV lysates and stocks isachieved.

In any of these embodiments, the host cell may stably express thoseadenoviral sequences that are deleted from the adenovirus comprising repand cap. For instance, a cell line such as 293 cells, which express E1,84-31 cells, which express E1 and E4 (Ref. 1), or 10-3 cells, whichexpress E1 and E4ORF6 (Ref. 11), may be used. Alternatively, a secondhelper virus is co-infected into the host cell and expresses thoseadenoviral sequences deleted from the adenovirus comprising rep and cap.For instance, if a second adenovirus comprising a transgene cassette isused to infect the host cell, this adenovirus could supply the deletedadenoviral sequences.

In another embodiment of this invention, the recombinant virus carryingthe rep gene may be any virus in which rep interferes with itsreplication. In this embodiment, the recombinant viral vector comprisesa rep gene in which the native p5 promoter of rep is removed andreplaced with a minimal promoter or with no promoter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Construction of recombinant shuttle plasmids pAV2cisEGFP,pAd-p5-RC and pAd-HSP-RC.

FIG. 2A. Genome of the parental E1/E3 deleted adenovirus.

FIG. 2B. Schematic diagrams of the recombinant adenoviruses Ad-p5-RC andAd-HSP-RC showing insertion of p5-rep-cap or HSP-rep-cap DNA sequencesinto the E1 locus of the parental viral genome to generate Ad-p5-RC orAd-HSP-RC, respectively.

FIG. 3A. Schematic diagram of the rep-cap insert in the E1 locus ofAd-p5-RC or Ad-HSP-RC showing the location of PCR primers relative tothe viral genome and expected PCR DNA products I, II, III, and IV. PCRanalysis is used to determine the integrity of AAV rep-cap DNA sequencesinserted into the adenovirus genome.

FIG. 3B. Ethidium bromide stained agarose gel of PCR products usingprimers whose locations are shown in FIG. 3A. Lanes 1, 3, 5, 7 are PCRproducts from viral DNA of Ad-p5-RC. Lanes 2, 4, 6, 8 are PCR productsfrom viral DNA of Ad-HSP-RC. M, 1 kb DNA ladder size marker (Gibco BRL).

FIG. 4. Production of rAAV after infection of 293-CG3 cells withAd-HSP-RC.

FIG. 5. Production of rAAV after co-infection of 293 cells withAd-HSP-RC and Ad-AAV-LacZ.

FIG. 6. Time-course study of rAAV production after co-infection of 293cells with Ad-HSP-RC and Ad-AAV-LacZ.

FIG. 7. Multiplicity of infection study of rAAV production afterco-infection of 293 cells with Ad-HSP-RC and Ad-AAV-LacZ.

FIG. 8. Ethidium bromide stained agarose gel of Hirt DNA fractionated todetect replicating rAAV DNA in 293 cells (lanes 1–5) or in control B50cells (lane 6). Lane 1, Ad-p5-RC infection of 293 cells; lane 2,Ad-HSP-RC infection of 293 cells; lane 3, Ad-AAVLacZ infection of 293cells; lane 4, Ad-HSP-RC and Ad-AAV-LacZ co-infection of 293 cells; lane5, Ad-p5-RC and Ad-AAV-LacZ co-infection of 293 cells; lane 6, sub100rand Ad-AAV-LacZ stepwise infection of B50 cells. M, 1 kb DNA Ladder sizemarker (Gibco BRL).

FIG. 9A. Ethidium bromide stained agarose gel of Hirt DNA samples from293 cells (lanes 2–6) or from control B-50 cells (lane 7) DNA samples.

FIG. 9B. Southern blot analysis of the gel shown in FIG. 9A hybridizedto a lacZ DNA probe. Lane 1, lacZ DNA fragment as a positive control;lane 2, Ad-HSP-RC infection of 293 cells; lane 3, Ad-p5-RC infection of293 cells; lane 4, Ad-AAVLacZ infection of 293 cells; lane 5, Ad-HSP-RCand Ad-AAV-LacZ co-infection of 293 cells; lane 6, Ad-p5-RC andAd-AAV-LacZ co-infection of 293 cells; lane 7, sub100r and Ad-AAV-LacZstepwise infection of B50 cells. M, 1 kb DNA Ladder size marker (GibcoBRL)

FIG. 10. Western blot analysis of Rep and Cap protein expression in 293cells infected with or Ad-p5-RC or Ad-HSP-RC viruses. Lane 1, Ad-p5-RCalone; lane 2, Ad-p5-RC+Ad-AAV-LacZ; lane 3, Ad-HSP-RC alone; lane 4,Ad-AAV-LacZ alone; lane 5, Ad-HSP-RC+Ad/AAV-LacZ.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention relates to a novel adenoviral vector and a methodfor producing high titer stocks of rAAV using this vector. Theadenoviral vector comprises a rep gene in which the native AAV p5promoter upstream of the rep coding sequences has been deleted oreffectively rendered inactive by mutation or partial deletion andreplaced by a minimal promoter or no promoter.

Although decreasing maximal production of Rep78 and Rep68 may increaserAAV production (see WO 97/06272 and WO 98/46728), there has been noevidence that one could obtain rAAV production if one replaced the p5promoter with either a minimal promoter that promotes only basalexpression of Rep78/68 or with no promoter, i.e., removing the promoteraltogether and incorporating rep into an adenovirus.

In a preferred embodiment, Rep78 and Rep68 are produced at much lowerlevels than Rep52 and Rep40 in 293 cells or other E1-complementing celllines infected with an adenovirus containing a minimal promoter drivingexpression of rep78 and rep68. See, e.g., Example 9 and FIG. 10. Inanother preferred embodiment, host cells are infected with an adenovirusvector comprising a rep gene that lacks any promoter. Although the exactamounts of Rep78 and Rep68 protein expressed in host cells infected byan adenovirus lacking any promoter upstream of rep coding sequences areunknown, it is expected that Rep78 and Rep68 protein levels would beexpressed from this recombinant adenovirus at much lower levels thanRep52 and Rep40 or at levels much lower than that expressed by wildtypeAAV during infection.

In one embodiment of the invention, the total amount of Rep78 and Rep68protein is less than 80%, more preferably less than 50%, of the totalamount of Rep52 and Rep40 produced in the infected cells. In a morepreferred embodiment, the total amount of Rep78 and Rep68 is less than25% of the total amount of Rep52 and Rep40 produced in the infectedcells. In an even more preferred embodiment, the total amount of Rep78and Rep68 is less than 15%, more preferably 10%, and more preferably isless than 5% of the total amount of Rep52 and Rep40 produced in theinfected cells. One may measure the amount of Rep proteins by any methodknown in the art. These methods include, without limitation,immunoprecipitation of metabolically labeled Rep proteins followed byseparation on SDS polyacrylamide gel electrophoresis and quantitation ofthe labeled protein, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), immunofluorescence of infected cells, andquantitative Western blot analysis using radioactive or enzymaticlabeling of an anti-Rep antibodies.

Experiments performed using an adenovirus vector comprising apromoterless rep gene demonstrate that the infected host cells alsoproduce rAAV. Without wishing to be bound by any theory, it is possiblethat in the absence of a promoter upstream of rep, low-leveltranscription of Rep78 and Rep68 may occur from the upstream ITR ofadenovirus or from sequences downstream of the ITR that are upstream ofthe rep gene.

The instant invention demonstrates that adenoviral vectors comprising arep gene with its native p5 promoter are unstable when infected intohost cells while adenoviral vectors comprising a rep gene with a minimalpromoter or no promoter are stably propagated in host cells. See Example4 and FIGS. 3A and 3B. Example 4 demonstrates that Ad-p5-RC, which is anadenovirus containing p5, rep and cap in the E1 site of the adenovirusvector, undergoes a rearrangement or deletion event in the rep-cap DNAsequences of the adenovirus vector when passaged in 293 cells. Incontrast, Ad-HSP-RC, which contains a minimal heat shock proteinpromoter (HSP), rep and cap, is stable after insertion into the E1 locusof the adenoviral genome and does not appear to undergo anyrearrangement when passaged in the same cells. In a preferredembodiment, a minimal promoter or promoterless rep-containing adenovirusof the instant invention is one which is stable upon propagation in adefined host cell system, such as 293 cells.

The deletion or rearrangement of rep and cap is further borne out byExamples 8–9 and FIGS. 8–10. Southern blot analysis demonstrates thatHirt DNA from 293 cells co-infected with Ad-HSP-RC and Ad-AAV-LacZ (anadenoviral vector in which a transgene cassette comprising lacZ flankedby AAV ITRs is inserted in E1 of adenovirus) contains LacZ DNA sequencesin AAV replicating form (RF) DNA, while Hirt DNA from 293 cellsco-infected with Ad-p5-RC and Ad-AAV-LacZ does not contain LacZ DNAsequences in AAV RF DNA. In addition, 293 cells express Rep and Capproteins when co-infected with Ad-HSP-RC and Ad-AAV-LacZ (see lane 5 ofFIG. 10), but do not express these proteins when co-infected withAd-p5-RC and Ad-AAV-LacZ (see lane 4 of FIG. 10).

The effect of the deletion or rearrangement in Ad-p5-RC is shown by thelevels of rAAV produced using this adenovirus vector. Little or noreplicating rAAV is produced in cells co-infected with Ad-p5-RC andAd-AAV-LacZ, while replicating rAAV is observed in 293 cells co-infectedwith Ad-HSP-RC and Ad-AAV-LacZ. See Example 8 and FIGS. 8, 9A and 9B.Similarly, sufficient amounts of replicating rAAV is produced in cellsthat have been infected with an adenovirus vector comprising repsequences downstream of no promoter.

Definitions and General Techniques

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. The practice of the presentinvention employs, unless otherwise indicated, conventional techniquesof chemistry, molecular biology, microbiology, recombinant DNA,genetics, virology and immunology. See, e.g., Sambrook et al., 1989,Ausubel et al., 1992, Harlow et al. 1989 (which are incorporated hereinby reference).

A “recombinant adeno-associated virus (rAAV) genome” comprises all or apart of an AAV genome, wherein the viral genome may be wild type or maycontain point mutations or deletions, and optionally comprises atransgene operably linked to expression control sequences. The transgenemay be regulated in cis or in trans. In a preferred embodiment, the rAAVgenome comprises a transgene flanked by AAV inverted terminal repeats(ITRs). The rAAV genome of the invention may be embedded in the genomeof an adenovirus vector to form a hybrid Ad/AAV. See U.S. Pat. No.5,586,152, herein incorporated by reference. Alternatively, the rAAVgenome may be introduced into a host cell by any route known in the art.The rAAV genome can be expressed transiently or stably in the host cell.

A “recombinant adeno-associated virus” or “rAAV” is the AAV derived fromthe rAAV genome described above. The rAAV preferably comprises atransgene. The rAAV comprising a transgene is capable of transducingmammalian cells and delivering the transgene thereto.

A “flanking element” or “flanking nucleic acid” is a nucleic acidsequence which, when located in positions flanking a transgene, permitsthe packaging of the transgene into an rAAV. The flanking elements ofAAV are inverted terminal repeats (ITRs). Flanking elements may be thenaturally-occurring ITRs from any one of AAV serotypes 1–6 or may beartificial nucleic acid elements, e.g. mutated sequences of ITRs, thathave the same or equivalent packaging function.

A “transgene” is a nucleic acid sequence that is to be delivered ortransferred to a mammalian cell. A transgene may encode a protein,peptide or polypeptide that is useful as a marker, reporter ortherapeutic molecule. The transgene also may be a selection gene, suchas one for antibiotic resistance. A transgene may also encode a protein,polypeptide or peptide that is useful for protein production, diagnosticassays or for any transient or stable gene transfer in vitro or in vivo.Alternatively, a transgene may not encode a protein but rather be usedas a sense or antisense molecule, ribozyme or other regulatory nucleicacid to modulate replication, transcription or translation of a nucleicacid to which it is complementary or to target a complementary mRNA fordegradation.

“Expression control sequences” are nucleic acid sequences that regulatethe expression of a gene by being operably linked to the gene ofinterest.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion.

A “transgene cassette” is a nucleic acid sequence comprising a transgeneoperably linked to expression control sequences in which the transgeneand expression control sequences are flanked by AAV flanking sequences.In a preferred embodiment, the flanking sequences are AAV ITRs.

An “adenovirus genome” is the nucleic acid molecule backbone of anadenovirus particle. The adenovirus genome may contain point mutations,deletions or insertions of nucleotides. The adenovirus genome mayfurther comprise a foreign gene.

An “adenovirus” is an encapsidated adenovirus genome capable of bindingto a mammalian cell and delivering the adenovirus genome to the cell'snucleus. The term “adenovirus” encompasses both recombinant andnon-recombinant adenoviruses. The term “adenovirus” also encompassesboth wildtype and mutant adenoviruses.

A “recombinant adenovirus” is an adenovirus which contains one or moregenes that are foreign to a wildtype adenovirus. Recombinantadenoviruses include, without limitation, those that include foreigngenes such as rep and/or cap, as well as adenoviruses that comprise anrAAV genome.

An “adenovirus vector” is a recombinant adenovirus comprising one ormore foreign genes, wherein the adenovirus vector is capable of bindingto a mammalian cell and delivering the foreign gene to the cell'snucleus. The foreign genes include, without limitation, genes such asrep and cap, rAAV genomes, such as transgenes and expression controlsequences, or any foreign gene that is useful in increasing productionof rAAV.

A “locus” is a site within a virus wherein a particular gene normallyresides. For instance, the “adenovirus E1 locus” is the site at which E1resides in adenovirus. If a foreign gene or nucleic acid is insertedinto a locus, it may either replace the gene that resides there or itmay be inserted at the site in addition to the gene that resides there.

An “AAV p5 promoter” or “p5 promoter” is one that is derived from anyAAV serotype, including AAV Serotypes 1 to 6, as well as any AAV thatinfects non-human species, such as avian and bovine AAV. The p5 promoterof AAV-2 directs the expression of rep78 and rep68, and is downregulatedby the Rep protein, and is upregulated by certain adenoviral proteins,including E1.

As used herein, the term “deleted p5 promoter” refers to a p5 promoterthat has been completely deleted from the AAV genome or to a p5 promoterthat has been effectively deleted or attenuated such that the p5promoter is less active when compared to the wildtype p5 promoter inpromoting transcription in a cell into which it has been introduced. Thep5 promoter may be effectively deleted or attenuated by any method,including, without limitation, removing or mutating a sufficient numberof nucleotides to render the p5 promoter less active or inactive ormoving the promoter relative to the coding sequences of rep such thatthe promoter is less active. In an alternative embodiment, one mayincrease the distance between the p5 promoter and the start codon of therep gene to decrease promoter activity. For instance, one may move thep5 promoter downstream of the rep gene or insert nucleotide sequencesbetween the p5 promoter and the downstream ATG start codon. One maymeasure whether a p5 promoter is effectively deleted by measuring thetranscription of a gene operably linked to the mutated or partiallydeleted p5 promoter and comparing the gene's transcription to thetranscription of a gene in a promoterless construct.

In a preferred embodiment, a p5 promoter is effectively deleted when itpromotes less than 80% of wildtype p5 promoter activity, more preferablyless than 50% of wildtype p5 promoter activity. In a more preferredembodiment, a p5 promoter is effectively deleted when it promotes lessthan 25% of wildtype p5 promoter activity. In an even more preferredembodiment, a p5 promoter is effectively deleted when it promotes lessthan 15%, more preferably promotes less than 10%, and more preferablyless than 5% of wildtype p5 promoter activity. In another preferredembodiment, a p5 promoter is effectively deleted when the rep gene towhich it is operably linked is not rearranged or deleted when anadenovirus comprising the effectively deleted p5 promoter and rep geneis infected into a host cell, such as 293 cells. In another preferredembodiment, a p5 promoter is effectively deleted when a host cellinfected with an adenovirus comprising rep and the deleted p5 promoterproduces rAAV at a high titer. In a preferred embodiment, the titer isat least 10² particles per cell; preferably at least 10³ particles percell; more preferably at least 10⁴ particles per cell; and, even morepreferably, at least 10⁵ or 10⁶ particles per cell. In general, thereare approximately 1×10³ to 3×10³ particles per transducing units (TU).The number of particles required to produce one TU varies based upon thetransgene, purification method and assay method.

A “minimal promoter” is one that essentially comprises only a TATA boxand promotes only very low or basal levels of transcription of rep78 andrep68. A promoter is a nucleotide sequence that promotes the initiationof transcription at a particular site by the cell's transcriptionalmachinery.

In a preferred embodiment, a minimal promoter promotes transcriptionthat is less than 80% of the wildtype p5 promoter, more preferably lessthan 50% of the wildtype p5 promoter, even more preferably less than 25%of the wildtype p5 promoter. In a more preferred embodiment, a minimalpromoter is one that promotes transcription that is less than 20% of thewildtype p5 promoter, even more preferably less than 15% of the wildtypep5 promoter. In an even more preferred embodiment, a minimal promoter isone that promotes transcription that is less than 10% of the wildtype p5promoter, even more preferably less than 5%, even more preferably lessthan 1% of the wildtype p5 promoter.

A minimal promoter also may be defined by functional measures. A minimalpromoter is one in which the rep gene to which it is operably linked isnot rearranged or deleted when an adenovirus comprising the minimalpromoter and rep gene is infected into a host cell, such as 293 cells. Aminimal promoter is one in which a host cell infected with an adenoviruscomprising a minimal promoter that regulates transcription of rep78 andrep68 produces rAAV at a high titer. In a preferred embodiment, thetiter is at least 10² particles per cell, preferably at least 10³particles per cell; more preferably at least 10⁴ particles per cell;and, even more preferably, at least 10⁵ or 10⁶ particles per cell.

Many minimal promoters are known in the art. Alternatively, anartificial minimal promoter may be constructed by using a sequence or aconsensus sequence of a TATA box and adding nucleotide sequences to the5′ and 3′ ends of the TATA box. The activity of the minimal promoter maybe measured by measuring the transcription of the artificial minimalpromoter and comparing it to an natural minimal promoter, such as theDrosophila heat shock protein promoter.

Rep78/68 is “promoterless” or has “no promoter” when the p5 promoter hasbeen deleted or effectively deleted, as defined supra, and no promoterhas been inserted in its place. Alternatively, rep78/68 is promoterlesswhen the p5 promoter has been deleted and is replaced by a heterologouspromoter that does not promote transcription in the host cell in whichthe adenovirus has been infected. For instance, rep78/68 would beconsidered promoterless if p5 were substituted by a promoter that wasactive in bacterial or insect cells, for example, but that was inactivein a mammalian host cell. In another embodiment, rep78/68 ispromoterless when the p5 promoter has been deleted and replaced by aninducible promoter that permits low-level expression of rep78/68.

The Adenoviral Vector

A large number of adenoviruses and adenoviral vectors are known,including human adenoviruses types 1–46, chimpanzee adenoviruses, canineadenoviruses, bovine adenoviruses [all available from the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209], and ovine adenoviruses (Both et al., WO 97/06826 A1). Anyof these adenoviruses may be used in this invention, provided that theadenovirus is able to infect the target host cell. For instance, a humanadenovirus would generally be used to infect a human cell, while abovine adenovirus would be used to infect a bovine cell.

In one embodiment, the adenoviral vector comprises the AAV rep genedownstream of a minimal promoter or no promoter (this is alternativelyreferred to as a minimal promoter or promoterless AAV rep gene), andsufficient helper virus functions for rAAV production in a host cell. Ina preferred embodiment, the adenoviral vector further comprises the AAVcap gene. The type of adenoviral sequences required for replication andencapsidation of the rAAV genome depends upon whether the host cellexpresses any helper functions or whether other vectors or viruses areintroduced into the host cell which express helper functions. Forinstance, if the adenovirus is to be used to infect a cell line thatexpresses E1, e.g., the 293 cell line, then the adenoviral vector couldcomprise rep and cap, and could also comprise those helper virusfunctions required in addition to E1 (e.g., E2a, E4ORF6 and VAI RNA). Ifthe adenovirus is used to infect a cell line such as 84-31, whichexpresses E1 and E4, then the adenoviral vector could express rep, cap,E2a and VAI RNA. If the adenovirus is used to infect a cell line thatdoes not express any helper functions, then the adenovirus vector couldcomprise, at least, E1 (both E1a and E1b) and E2a, and, optionally, maycomprise E4ORF6 and VAI RNA. In an alternative embodiment, helperfunctions may be supplied by chemical or physical methods or by otherhelper viruses.

The recombinant adenovirus comprising the rep gene downstream of aminimal promoter or no promoter may be produced by any method known inthe art. In one preferred method, the recombinant adenovirus of theinstant invention is produced using homologous recombination. In anotherpreferred method, the recombinant adenovirus is produced using Cre-loxrecombination (12).

In an alternative embodiment, some or all of the helper virus functionsmay be provided by nucleic acid sequences that are introduced into thehost cell. For instance, the host cell may be co-infected with a secondvirus, such as an adenovirus, that expresses some or all of the requiredhelper functions. In a preferred embodiment, a second adenoviral vectorcomprises a transgene cassette and further comprises helper functionsthat are not expressed by either the host cell or the adenoviruscomprising the rep gene. See, e.g., Examples 6–8. Alternatively, some orall of the helper virus functions may be provided by any method known inthe art, such as by transfection or direct injection, as discussedabove.

Based on this description, other embodiments of the adenoviral vectorwill be readily apparent to those of ordinary skill in the art. Otherviral vectors in which Rep interferes with viral replication also may beused.

Rep and Cap Nucleic Acids

In AAV's life cycle, both rep and cap are required for excision,replication and encapsidation of the recombinant viral genome into aninfectious recombinant vector or virus. The Rep and Cap proteins mayhave a naturally occurring sequence derived from any serotype of AAV,including serotypes 1 to 6. In one preferred embodiment, the rep and capgenes are derived from the same AAV serotype. In another preferredembodiment, the rep and cap genes are derived from different AAVserotypes to permit the production a pseudotyped rAAV. Pseudotyped rAAVis desirable in cases in which the rAAV is to be administered to apatient as a gene therapy vector and there are existing neutralizingantibodies in the patient's serum to the capsid proteins of one AAVserotype and not to another AAV serotype. The cap gene from the serotypeto which there is an antibody response may be exchanged by the cap genefrom a different serotype of AAV to which there is no antibody response.For example, the rep gene from AAV-2 may be used with the cap gene fromAAV-1 to produce a pseudotyped rAAV-2, or vice-versa.

In an alternative embodiment, the Rep and/or Cap proteins may have amutated sequence, including insertions, deletions, fragments or pointmutations of particular amino acid residues, so long as the mutated Repand/or Cap proteins retain their respective excision, replication andencapsidation functions. In a preferred embodiment, thenaturally-occurring Rep and Cap amino acid sequences from AAV-2 areused. In one embodiment, a single adenovirus comprises the nucleic acidsequences encoding the Rep and Cap proteins. In another embodiment, thenucleic acid sequences encoding the Rep and Cap proteins are inserted ata single site within one adenovirus, e.g., both rep and cap are insertedat E1, E3 or E4 of adenovirus. Alternatively, the nucleic acid sequencesencoding Rep and Cap, respectively, are each inserted at different lociin the adenovirus genome, e.g., the nucleic acid sequence encoding Repmay be in E1, and the nucleic acid sequence encoding Cap may be in E4,and other combinations thereof. Alternatively, a cell line comprisingthe cap gene may be used, in which case only the rep gene would berequired on the adenoviral vector. See, e.g., WO 98/27204.

The minimal promoter that regulates expression of Rep78 or Rep68 may beany promoter that promotes only basal expression of the rep gene in ahost cell. In general, the promoter is one that essentially contains aTATA box as its only regulatory element. In a preferred embodiment, theminimal promoter is the Drosophila heat shock promoter (HSP). In anotherpreferred embodiment, the minimal promoter is the minimal promoterderived from the adenovirus E1b gene that provides only basal promoteractivity. In another preferred embodiment, the minimal promoter is a 70nucleotide DNA element derived from the promoter region upstream of theadenovirus pIX gene. The minimal pIX promoter comprises a TATA box andan Sp1 box, and, in Ad5, corresponds to nucleotides 3511 to 3580. Manyother adenovirus serotypes contain the pIX gene and its upstreampromoter as well, and the minimal promoters derived from these pIXpromoters are encompassed by this invention as well. Other minimalpromoters are well known in the art and may be used in the practice ofthis invention. In another preferred embodiment, the p5 promoter isdeleted altogether and replaced by no promoter at all.

In another embodiment of the invention, the activity of the Rep78/68proteins are attenuated by mutating the rep78/68 genes to produceRep78/68 proteins that are less active than wildtype Rep78/68. This maybe done by altering the coding sequence of the Rep78/68 proteins to makethem less active. Alternatively, one may alter the DNA sequence ofrep78/68 to destabilize the RNA encoded by the gene and thus decreasethe amount of Rep78/68 proteins produced.

One may determine whether a DNA sequence is appropriate for use as aminimal promoter by inserting the DNA sequence upstream of the rep78/68ATG codon in a plasmid construct or an adenoviral vector andtransfecting or infecting, respectively, a host cell that comprises anrAAV genome, incubating the host cell under conditions in which rAAV isproduced, measuring the titer of rAAV produced, and comparing the titerto that produced using a control plasmid or adenovirus comprising a repgene whose expression is regulated by a minimal promoter. The host cellmay comprise the rAAV genome stably or transiently. Alternatively, onemay measure the level of Rep78 and Rep68 produced in the host cell afterinfection to determine if sufficiently low levels of Rep78 and Rep68 areproduced.

Helper Functions

As discussed above, AAV requires helper functions for excision,replication and encapsidation of AAV. AAV helper functions can beprovided by adenovirus, herpesvirus [including herpes simplex virus type1 (HSV-1) or type 2 (HSV-2), cytomegalovirus (CMV) and pseudorabiesvirus (PRV)] or by exposure of the cells to different chemical orphysical agents. Alternatively, one of skill in the art may determinewhich helper functions are required by producing rAAV using thecompositions and methods disclosed in the instant specification.

To identify which helper functions are required for high levels of rAAVproduction, one may transfect a host cell containing an rAAV genome witha plasmid comprising rep and cap and then transfect with one or nucleicacids encoding various potential helper functions to determine whichpotential helper functions are required for rAAV production. The rAAVgenome may be stably integrated into the host cell or may be transfectedor infected into the host cell by methods known in the art. Aftertransfecting the host cell with the nucleic acid encoding the potentialhelper function, one may then measure the titer of the rAAV that isproduced to determine if the nucleic acid encodes a helper function.

In a preferred embodiment, the helper functions are nucleic acidsderived from a virus. In a more preferred embodiment, the helperfunctions are derived from adenovirus types 2 or 5, HSV-1, HSV-2, CMV orPRV. In an even more preferred embodiment, the helper functions are E1a,E1b, E2a, E4ORF6 proteins and VAI RNA from adenovirus. In anotherpreferred embodiment, the nucleic acid encodes the helper functions fromthe helicase-primase complex of HSV (UL5, UL8 and UL52) and the majorsingle-stranded DNA binding protein of HSV (UL29). Alternatively, helperfunctions for recombinant AAV may be provided by chemical or physicalagents, including ultraviolet light, cycloheximide, hydroxyurea andvarious carcinogens.

The required helper functions for production of a rAAV may be deliveredto the host cell by any method known in art. The helper functions may bedelivered by transfection with a vector, such as a plasmid, by infectionwith a viral vector comprising the helper functions, or by any othermethod known in the art, including those discussed above (e.g.,biolistic injection of DNA, use of DNA conjugates, etc.). Thetransfection or infection may be stable or transient. Alternatively, thecell line may stably express (either on an extrachromosomal episome orthrough integration in the cell's genome) the helper functions. Inaddition, some of the helper functions may be expressed by the mammaliancell line while other helper functions are introduced by a vector. Thus,for production of rAAV in 293 cells (ATCC CRL-1573), whichconstitutively produce adenoviral E1a and E1b proteins, only E2a, E4ORF6and VAI must be introduced into the host cell by transfection orinfection of a vector.

In a preferred embodiment, the helper functions are transduced into thehost cells by an adenovirus. In a more preferred embodiment, some or allof the helper functions are transduced into the host cell by theadenovirus that comprises the rep and/or cap genes. In a preferredembodiment, the native helper function sequences are used. However,mutated helper function sequences may be used so long as they retaintheir helper function activity. The helper function nucleic acids may besupplied with its native promoter or may be under the regulatory controlof a variety of promoters, constitutive or inducible, such as the CMVimmediate-early promoter/enhancer or the zinc-inducible metallothioneinpromoter, respectively, as known in the art or as described above.

The rAAV Transgene Cassette

In order to manufacture a rAAV containing a transgene, the method of thepresent invention begins with a desired transgene, then associates thetransgene with appropriate expression regulatory sequences (ERS), e.g.,promoter, enhancer, polyadenylation site, then inserts thisERS-transgene construct between AAV flanking sequences, e.g., the ITRs,in place of rep and cap genes normally found therein. Where the lengthof the ERS-transgene cassette is shorter than the AAV rep and capsequences, and that shorter length would pose an obstacle to properpackaging, an optional spacer or “stuffer” sequence may be inserted inorder to maintain the proper length for packaging. The transgenecassette comprised of the ERS-transgene bordered by the AAV flankingsequences may then be embedded in an adenovirus vector separate fromthat which carries the rep gene. Alternatively, the transgene cassettemay be inserted into a plasmid vector and transfected into a host cell.The transgene cassette may be maintained in the host cell stably, eitherby integration into the host cell genome or as an episome, or may beintroduced transiently, such as by infection with a hybrid Ad/AAV virus.See, e.g., Examples 3 and 6–8. Each element of the transgene cassette isfurther described below:

The Transgene

A transgene is a nucleic acid encoding a protein of interest; it may bea gene to allow for genetic or drug selection, e.g., a gene conferringresistance to antibiotics, or a reporter gene allowing detection, e.g.,by color in the case of the use of green fluorescent protein.Alternatively, the transgene may be one that is useful for correctiveapplications. For instance, a transgene may be a normal gene thatreplaces or augments the function of a patient's defective gene. Thetransgene may be one that counteracts the effects of a disease, such asintroduction and expression of a gene that is distinct from the one thatit replaces or augments, but which has the same function or compensatesfor the defective gene's function. The transgene may be a gene whichblocks or represses the expression of a malfunctioning, mutated, orviral gene in the patient, thereby giving rise to a corrective effect. Atransgene may also be a protective gene, such as one that preventscellular apoptosis, injury, toxicity or death. A transgene may also beused for immunization against various agents, by provoking animmunogenic response in an animal. Delivery of therapeutic transgenes toa patient thus effects some level of correction of a defect or isbeneficial for prevention of disease. The transgene also may be a genewhich would confer sensitivity to a reagent that results in cellulartoxicity, e.g., introduction of HSV thymidine kinase, which conferssensitivity to gancyclovir. The transgene also may be one which isuseful for production of proteins in vitro, such as for large-scaleproduction of therapeutic proteins.

Many gene therapy methods involve supplying an exogenous gene toovercome a deficiency in the expression of a gene in a patient. Some ofthese deficiencies are congenital and are due to a mutation in aparticular gene in all the cells of the patient. For instance, in cysticfibrosis, there are one or more mutations in the gene encoding thecystic fibrosis transmembrane conductance regulator (CFTR) whichprevents the CFTR protein from functioning properly. In other cases, adeficiency in gene expression is due to an accident or disease thatoccurs during the patient's life. For instance, in Type I diabetesmellitus, the β pancreatic islet cells, which produce insulin, aredestroyed, such that patients with this disease can no longer synthesizeinsulin. In other cases, the endogenous gene may be structurally normalbut is not transcribed and/or translated in high enough quantities dueto disease, medical treatment or other environmental conditions, ormutations in the regulatory elements of the endogenous gene. Forexample, there are a number of blood disorders, such as anemia, in whichthere is insufficient production of red blood cells, which may betreated with erythropoietin (EPO) or with a transgene encoding EPO.Conversely, gene therapy methods may be used where overexpression of aparticular gene results in a disease state. For instance, overexpressionof c-myc by the immunoglobulin heavy chain promoter results in leukemia.Transgenes may also be used for genetic immunization, i.e., to elicit animmune response to a pathogen in an animal, including humans. Forinstance, a transgene may include a sequence from a viral, bacterial orfungal pathogen, such as influenza virus, human immunodeficiency virus(HIV), or mycobacterium tuberculosis.

Appropriate genes for expression in the cell include, withoutlimitation, those genes which are normally expressed in cells but whoseproducts are produced in abnormal amounts due to over- orunder-expression. Alternatively, the appropriate gene for expression isone which expresses a normal gene product which replaces a defectivegene product, encodes ribozymes or antisense molecules which repair ordestroy mutant cellular RNAs expressed from mutated genes, or modifiesor destroys viral RNAs. Transgenes used for production of proteins invitro include proteins such as secreted factors, including hormones,growth factors and enzymes.

The composition of the transgene sequence depends upon the intended usefor the resulting rAAV. For example, one type of transgene sequencecomprises a reporter or marker sequence, which upon expression producesa detectable signal. Such reporter or marker sequences include, withoutlimitation, DNA sequences encoding E. coli β-lactamase, β-galactosidase(LacZ), alkaline phosphatase, HSV thymidine kinase, green fluorescentprotein (GFP), bacterial chloramphenicol acetyltransferase (CAT),firefly luciferase, eukaryotic membrane bound proteins including, forexample, CD2, CD4, CD8, the influenza hemagglutinin protein, and otherswell known in the art, to which high affinity antibodies directed tothem exist or can be made routinely, and fusion proteins comprising amembrane bound protein appropriately fused to an antigen tag domainfrom, among others, hemagglutinin or myc.

These sequences, when associated with regulatory elements which drivetheir expression, provide signals detectable by conventional means,including enzymatic, radiographic, calorimetric, fluorescence or otherspectroscopic assays, fluorescent activated cell sorting assay andimmunological assays, including ELISA, RIA and immunohistochemistry. Forexample, where the transgene is the LacZ gene, the presence of a rAAV isdetected by assays for β-galactosidase activity. Similarly, where thetransgene is luciferase, the rAAV gene expression may be measured bylight production in a luminometer.

However, desirably, the transgene is a non-marker gene which can bedelivered to a cell or an animal via the rAAV produced by this method.The transgene may be selected from a wide variety of gene productsuseful in biology and medicine, such as proteins, sense or antisensenucleic acids (e.g., RNAs), or catalytic RNAs. The invention may be usedto correct or ameliorate gene deficiencies, wherein normal genes areexpressed but at greater than normal or less than normal levels, and mayalso be used to correct or ameliorate genetic defects wherein afunctional gene product is not expressed. A preferred type of transgenesequence is a therapeutic gene which expresses a desired corrective geneproduct in a host cell at a level sufficient to ameliorate the disease,including partial amelioration. These therapeutic nucleic acid sequencestypically encode products which, upon expression, are able to correct,complement or compensate an inherited or non-inherited genetic defect,or treat an epigenetic disorder or disease. However, the selectedtransgene may encode any product desirable for study. The selection ofthe transgene sequence is not a limitation of this invention. Choice ofa transgene sequence is within the skill of the artisan in accordancewith the teachings of this application.

The invention also includes methods of producing rAAV and compositionsthereof which can be used to correct or ameliorate a gene defect causedby a multi-subunit protein. In certain situations, a different transgenemay be used to encode each subunit of the protein. This may be desirablewhen the size of the DNA encoding the protein subunit is large, e.g.,for an immunoglobulin or the platelet-derived growth factor receptor. Inorder for the cell to produce the multi-subunit protein, a cell would beinfected with rAAV expressing each of the different subunits.

Alternatively and more preferably, different subunits of a protein maybe encoded by the same transgene. In this case, a single transgene wouldinclude the DNA encoding each of the subunits, with the DNA for eachsubunit separated by an internal ribosome entry site (IRES). The use ofIRES permits the creation of multigene or polycistronic mRNAs. IRESelements are able to bypass the ribosome scanning model of 5′ methylatedcap-dependent translation and begin translation at internal sites(Pelletier and Sonenberg, 1988). For example, IRES elements fromhepatitis C and members of the picornavirus family (e.g., polio andencephalomyocarditis) have been described, as well an IRES from amammalian mRNA (Macejak and Sarnow, 1991). IRES elements can be linkedto heterologous open reading frames. By virtue of the IRES element, eachopen reading frame is accessible to ribosomes for efficient translation.Thus, multiple genes can be efficiently expressed using a singlepromoter/enhancer to transcribe a single message. This is preferred whenthe size of the DNA encoding each of the subunits is sufficiently smallthat the total of the DNA encoding the subunits and the IRES is nogreater than the maximum size of the DNA insert that the virus canencompass. For instance, for rAAV, the insert size can be no greaterthan approximately 4.8 kilobases; however, for an adenovirus which lacksall of its helper functions, the insert size is approximately 28kilobases.

Useful gene products include hormones and growth and differentiationfactors including, without limitation, insulin, glucagon, growth hormone(GH), parathyroid hormone (PTH), calcitonin, growth hormone releasingfactor (GRF), thyroid stimulating hormone (TSH), adrenocorticotropichormone (ACTH), prolactin, melatonin, vasopressin, β-endorphin,met-enkephalin, leu-enkephalin, prolactin-releasing factor,prolactin-inhibiting factor, corticotropin-releasing hormone,thyrotropin-releasing hormone (TRH), follicle stimulating hormone (FSH),luteinizing hormone (LH), chorionic gonadotropin (CG), vascularendothelial growth factor (VEGF), angiopoietins, angiostatin,endostatin, granulocyte colony stimulating factor (GCSF), erythropoietin(EPO), connective tissue growth factor (CTGF), basic fibroblast growthfactor (bFGF), bFGF2, acidic fibroblast growth factor (aFGF), epidermalgrowth factor (EGF), transforming growth factor α (TGFα),platelet-derived growth factor (PDGF), insulin-like growth factors I andII (IGF-I and IGF-II), any one of the transforming growth factor β(TGFβ) superfamily comprising TGFβ, activins, inhibins, or any of thebone morphogenic proteins (BMP) BMPs 1–15, any one of theheregulin/neuregulin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3, NT-4/5 and NT-6, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurtuin, persephin, agrin, any one of the family ofsemaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor(HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful gene products include proteins that regulate the immunesystem including, without limitation, cytokines and lymphokines such asthrombopoietin (TPO), interleukins (IL) IL-1α, IL-1β, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, and IL-17, monocyte chemoattractant protein (MCP-1), leukemiainhibitory factor (LIF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocytecolony stimulating factor (M-CSF), Fas ligand, tumor necrosis factors αand β (TNFα and TNFβ), interferons (IFN) IFN-α, IFN-β and IFN-γ, stemcell factor, flk-2/flt3 ligand. Gene products produced by the immunesystem are also encompassed by this invention. These include, withoutlimitations, immunglobulins IgG, IgM, IgA, IgD and IgE, chimericimmunoglobulins, humanized antibodies, single chain antibodies, T cellreceptors, chimeric T cell receptors, single chain T cell receptors,class I and class II MHC molecules, as well as engineered MHC moleculesincluding single chain MHC molecules. Useful gene products also includecomplement regulatory proteins such as membrane cofactor protein (MCP),decay accelerating factor (DAF), CR1, CR2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. Examples of such receptors includeflt-1, flk-1, TIE-2; the trk family of receptors such as TrkA, MuSK,Eph, PDGF receptor, EGF receptor, HER2, insulin receptor, IGF-1receptor, the FGF family of receptors, the TGFβ receptors, theinterleukin receptors, the interferon receptors, serotonin receptors,α-adrenergic receptors, β-adrenergic receptors, the GDNF receptor, p75neurotrophin receptor, among others. The invention encompasses receptorsfor extracellular matrix proteins, such as integrins, counter-receptorsfor transmembrane-bound proteins, such as intercellular adhesionmolecules (ICAM-1, ICAM-2, ICAM-3 and ICAM-4), vascular cell adhesionmolecules (VCAM), and selectins E-selectin, P-selectin and L-selectin.The invention encompasses receptors for cholesterol regulation,including the LDL receptor, HDL receptor, VLDL receptor, and thescavenger receptor. The inventions encompasses the apolipoproteinligands for these receptors, including ApoAI, ApoAIV and ApoE. Theinvention also encompasses gene products such as steroid hormonereceptor superfamily including glucocorticoid receptors and estrogenreceptors, Vitamin D receptors and other nuclear receptors. In addition,useful gene products include antimicrobial peptides such as defensinsand maginins, transcription factors such as jun, fos, max, mad, serumresponse factor (SRF), AP-1, AP-2, myb, MRG1, CREM, Alx4, FREAC1, NF-κB,members of the leucine zipper family, C2H4 zinc finger proteins,including Zif268, EGR1, EGR2, C6 zinc finger proteins, including theglucocorticoid and estrogen receptors, POU domain proteins, exemplifiedby Pit 1, homeodomain proteins, including HOX-1, basic helix-loop-helixproteins, including myc, MyoD and myogenin, ETS-box containing proteins,TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1,CCAAT-box binding proteins, interferon regulation factor 1 (IRF-1),Wilms tumor protein, ETS-binding protein, STAT, GATA-box bindingproteins, e.g., GATA-3, and the forkhead family of winged helixproteins.

Other useful gene products include carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,factor VII, factor VIII, factor IX, factor II, factor V, factor X,factor XII, factor XI, von Willebrand factor, superoxide dismutase,glutathione peroxidase and reductase, heme oxygenase, angiotensinconverting enzyme, endothelin-1, atrial natriuetic peptide,pro-urokinase, urokinase, plasminogen activator, heparin cofactor II,activated protein C (Factor V Leiden), Protein C, antithrombin,cystathione beta-synthase, branched chain ketoacid decarboxylase,albumin, isovaleryl-CoA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase (also referred to asP-protein), H-protein, T-protein, Menkes disease protein, tumorsuppressors (e.g., p53), cystic fibrosis transmembrane regulator (CFTR),the product of Wilson's disease gene PWD, Cu/Zn superoxide dismutase,aromatic aminoacid decarboxylase, tyrosine hydroxylase, acetylcholinesynthetase, prohormone convertases, protease inhibitors, lactase,lipase, trypsin, gastrointestinal enzymes including chyromotrypsin, andpepsin, adenosine deaminase, α1 anti-trypsin, tissue inhibitor ofmetalloproteinases (TIMP), GLUT-1, GLUT-2, trehalose phosphate synthase,hexokinases I, II and III, glucokinase, any one or more of theindividual chains or types of collagen, elastin, fibronectin,thrombospondin, vitronectin and tenascin, and suicide genes such asthymidine kinase and cytosine deaminase. Other useful proteins includethose involved in lysosomal storage disorders, including acidβ-glucosidase, α-galactosidase a, α-1-iduronidase, iduroate sulfatase,lysosomal acid α-glucosidase, sphingomyelinase, hexosamina\idase A,hexomimidases A and B, arylsulfatase A, acid lipase, acid ceramidase,galactosylceramidase, α-fucosidase, α-, β-mannosidosis,aspartylglucosaminidase, neuramidase, galactosylceramidase,heparan-N-sulfatase, N-acetyl-α-glucosaminidase, Acetyl-CoA:α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfatesulfatase, N-acetylgalactosamine-6-sulfate sulfatase, arylsulfatase B,β-glucuoronidase and hexosaminidases A and B.

Other useful transgenes include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides or polypeptides having anon-naturally occurring amino acid sequence containing insertions,deletions or amino acid substitutions. For example, single-chainengineered immunoglobulins could be useful in certain immunocompromisedpatients. Other useful proteins include truncated receptors which lacktheir transmembrane and cytoplasmic domain. These truncated receptorscan be used to antagonize the function of their respective ligands bybinding to them without concomitant signaling by the receptor. Othertypes of non-naturally occurring gene sequences include sense andantisense molecules and catalytic nucleic acids, such as ribozymes,which could be used to modulate expression of a gene.

Other useful transgenes include those that encode antigenic peptidescapable of generating an immune response. Recombinant vectors comprisingthese transgenes can be used for genetic immunization. Useful transgenesinclude those that encode peptides specific for Epstein Barr virus; HIV;simian immunodeficiency virus (SIV); human T-cell leukemia viruses I andII (HTLV-I and HTLV-II); hepatitis A, B, C, D, E and SEN; pseudorabiesvirus; rabies virus; cytomegalovirus; respiratory syncytial virus;parainfluenza virus types 1–4; mumps virus; rubella virus; polio virus;measles virus; influenza virus types A, B and C; rotavirus; herpessimplex viruses types 1 and 2; varicella-zoster virus; human herpesvirus type 6, 7 and 8; hantavirus; denguevirus, sindbisvirus,adenoviruses; chlamydia pneumoniae; chlamydia trachomatis; mycoplasmapneumoniae; mycobacterium tuberculosis; atypical mycobacteria; felineleukemia virus; feline immunodeficiency virus; bovine immunodeficiencyvirus; equine infectious anemia virus; caprine arthritis encephalitisvirus; visna virus; Staphlococcus species and Streptococcus species. Thetransgenes may also be directed against peptides from tumor antigens toprovide immunization for tumors and cancers.

Expression Control Sequences

A great number of expression control sequences—native, constitutive,inducible and/or tissue-specific—are known in the art and may beutilized to drive expression of the transgene and the nucleic acidsequences encoding the replication and encapsidation functions of therAAV, the helper functions and the ligand. The choice of expressioncontrol sequence depends upon the type of expression desired. Foreukaryotic cells, expression control sequences typically include apromoter, an enhancer, such as one derived from an immunoglobulin gene,SV40, cytomegalovirus, etc., and a polyadenylation sequence. Thepolyadenylation sequence generally is inserted following the transgenesequences and before the 3′ flanking sequence of the transgene. Atransgene-carrying molecule useful in the present invention may alsocontain an intron, desirably located between the promoter/enhancersequence and the transgene. One possible intron sequence is also derivedfrom SV40 and is referred to as the late16S/19S intron. Another vectorelement that may be used is an internal ribosome entry site (IRES), asdescribed above. An IRES element is used to produce more than onepolypeptide from a single transcript. An IRES element can be used forthe transgene or for any of the other nucleic acid sequences encodingthe replication and encapsidation polypeptides, the helper functions orthe ligand. Selection of these and other common vector elements areconventional and many such sequences are available [see, e.g., Sambrooket al, and references cited therein at, for example, pages 3.18–3.26 and16.17–16.27 and Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, New York, 1989].

In one embodiment, high-level constitutive expression will be desired.Examples of such promoters include, without limitation, the retroviralRous sarcoma virus (RSV) LTR promoter/enhancer, the cytomegalovirus(CMV) immediate early promoter/enhancer [see, e.g., Boshart et al, Cell,41:521–530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the cytoplasmic β-actin promoter and the phosphoglycerolkinase (PGK) promoter.

In another embodiment, inducible promoters may be desired. Induciblepromoters are those which are regulated by exogenously suppliedcompounds, either in cis or in trans, including without limitation, thezinc-inducible metallothionine (MT) promoter; the dexamethasone(Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7polymerase promoter system [WO 98/10088]; the ecdysone insect promoter[No et al, Proc. Natl. Acad. Sci. USA, 93:3346–3351 (1996)]; thetetracycline-repressible system [Gossen et al, Proc. Natl. Acad. Sci.USA, 89:5547–5551 (1992)]; the tetracycline-inducible system [Gossen etal., Science, 268:1766–1769 (1995); see also Harvey et al., Curr. Opin.Chem. Biol., 2:512–518 (1998)]; the RU486-inducible system [Wang et al.,Nat. Biotech., 15:239–243 (1997) and Wang et al., Gene Ther. 4:432–441(1997)]; and the rapamycin-inducible system [Magari et al., J. Clin.Invest., 100:2865–2872 (1997); Rivera et al., Nat. Medicine, 2:1028–1032(1996)]. Other types of inducible promoters which may be useful in thiscontext are those which are regulated by a specific physiological state,e.g., temperature, acute phase, or in replicating cells only.

In another embodiment, the native promoter for the transgene or nucleicacid sequence of interest will be used. The native promoter may bepreferred when it is desired that expression of the transgene or thenucleic acid sequence should mimic the native expression. The nativepromoter may be used when expression of the transgene or other nucleicacid sequence must be regulated temporally or developmentally, or in atissue-specific manner, or in response to specific transcriptionalstimuli. In a further embodiment, other native expression controlelements, such as enhancer elements, polyadenylation sites or Kozakconsensus sequences may also be used to mimic the native expression.

In one embodiment, the recombinant viral genome comprises a transgeneoperably linked to a tissue-specific promoter. For instance, ifexpression in skeletal muscle is desired, a promoter active in musclemay be used. These include the promoters from genes encoding skeletalα-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, aswell as synthetic muscle promoters with activities higher thannaturally-occurring promoters [see Li et al., Nat. Biotech., 17:241–245(1999)]. Examples of promoters that are tissue-specific are known forliver [albumin, Miyatake et al. J. Virol., 71:5124–32 (1997); hepatitisB virus core promoter, Sandig et al., Gene Ther., 3:1002–9 (1996);alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503–14(1996)], bone [osteocalcin, Stein et al., Mol. Biol. Rep., 24:185–96(1997); bone sialoprotein, Chen et al., J. Bone Miner. Res., 11:654–64(1996)], lymphocytes [CD2, Hansal et al., J. Immunol., 161:1063–8(1998); immunoglobulin heavy chain; T cell receptor a chain], neuronal[neuron-specific enolase (NSE) promoter, Andersen et al. Cell. Mol.Neurobiol. 13:503–15 (1993); neurofilament light-chain gene, Piccioli etal., Proc. Natl. Acad. Sci. USA, 88:5611–5 (1991); the neuron-specificvgf gene, Piccioli et al., Neuron. 15:373–84 (1995)]; among others.

Of course, not all vectors and expression control sequences willfunction equally well to express all of the transgenes or other nucleicacid sequences of this invention. However, one of skill in the art maymake a selection among these expression control sequences withoutdeparting from the scope of this invention. Suitable promoter/enhancersequences which function in the appropriate host cell of choice may beselected by one of skill in the art using the guidance provided by thisapplication. Such selection is a routine matter and is not a limitationof the molecule or construct.

One may identify a suitable expression control sequence for a desiredtransgene by selecting one or more expression control sequences andoperably linking the expression control sequence to the nucleic acidsequence to be regulated. Then, one may insert these operably linkedsequences comprising the expression control sequence and regulatedsequence into the genome of the adenovirus vector. In one embodiment,one may insert a recombinant viral genome comprising the expressioncontrol sequence and the transgene into a vector of the instantinvention. After following one of the methods for producing andpackaging the rAAV as taught in this specification one may infectsuitable cells in vitro or in vivo. The number of copies of thetransgene in the cell may be monitored by Southern blotting orquantitative PCR; the level of RNA expression may be monitored byNorthern blotting or quantitative RT-PCR; and the level of proteinexpression may be monitored by Western blotting, immunohistochemistry,ELISA, RIA, tests of the transgene's gene product's biological activity,either in vitro or in vivo, or tests for correction or amelioration of agenetic defect.

Flanking Elements

The naturally-occurring AAV ITRs consist of repeated sequences, usuallybut not necessarily approximately 145 nucleotides in length, at the 5′and 3′ ends of the AAV genome. The AAV ITRs are required forreplication, excision and encapsidation of both wild type andrecombinant AAV virions. The ITRs flank the transgene when the AAV DNAintegrates into a host cell chromosome. When rAAV is rescued from thehost chromosome, the ITRs excise along with the transgene and remain inflanking positions surrounding the rescued DNA, in a form suitable forpackaging into virions. The ITRs may be derived from any one of theadeno-associated viruses known, including AAV serotypes 1 to 6. In apreferred embodiment of the invention, the rAAV comprises a selectedtransgene operably linked to expression regulatory sequences and AAVflanking elements.

Host Cells

Any type of mammalian cell that can be adapted to cell culture may beused as a host cell to produce the recombinant viral genome. In general,a host cell used in this invention is one that may be infected by theadenovirus vector of the instant invention. Another preferredcharacteristic of the host cell is that it is able to replicate the rAAVat high levels.

Appropriate host cells include, without limitation, CHO, BHK, MDCK andvarious murine cells, e.g., 10T1/2 and WEHI cells, African green monkeycells such as VERO, COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and humancells such as WI38, MRC5, A549, human embryonic retinoblast (HER), humanembryonic kidney (HEK), human embryonic lung (HEL) and HT1080 cells. Ina preferred embodiment, appropriate cells include 293 cells (humanembryonic kidney cells that express adenoviral E1a and E1b proteins),911 or PER.C6 cells (human embryonic retinoblast cells that expressadenoviral E1; see WO 97/00326), B50 cells (HeLa cells that express AAVrep and cap, see PCT US98/19463), 84-31 cells (293-based cells thatexpress adenovirus E1a, E1b and E4, Ref. 4), 10-3 cells (293-based cellsthat express adenovirus E1a, E1b and E4ORF6, Ref. 11), 3T3 cells (mouseembryonic fibroblast cell line), NIH3T3 cells (subline of 3T3 cells),HepG2 cells (human liver carcinoma cell line), Saos-2 cells (humanosteogenic sarcoma cell line), HuH7 cells or HeLa cells (human carcinomacell line).

In addition to the host cells listed above, other host cells may beused. One may determine whether a cell line would be suited for use as amammalian host cell by infecting the cell line with an adenovirus of theinstant invention in the presence of rep, cap and all required helperfunctions, culturing the cells under conditions in which rAAV isproduced, and then measuring the titer of infectious rAAV. One may thencompare the titer of infectious rAAV produced in the potential host cellwith the titers produced by other host cells to determine whether thecell line is good for rAAV production.

Methods of Producing Recombinant Adeno-Associated Virus from Adenovirus

Another aspect of the instant invention is a method of producing rAAVfrom the adenovirus of the instant invention. The method comprises thesteps of:

1. Infecting host cells comprising an rAAV genome with an adenoviruscomprising a rep gene under the regulatory control of a minimal promoteror no promoter;

2. growing the infected host cells under conditions in which the rAAVgenome is excised, replicated and encapsidated; and

3. collecting the rAAV from the mammalian host cells.

The host cells may be any mammalian cell known in the art or asdescribed herein. The host cell, prior to infection by the adenoviruscomprising the rep gene, may be one that expresses one or more of thefollowing genetic elements: 1) the cap gene, 2) some or all necessaryhelper functions (e.g., 293 cells), and/or 3) an rAAV genome.Alternatively, the host cell may comprise none of these genetic elementsprior to infection by the adenovirus. In this case, the genetic elementsare supplied by the adenovirus comprising the rep gene, other viralvectors, and/or plasmids.

The host cells may be infected by the adenovirus by any method known inthe art or as described herein. Methods for infecting host cells withadenoviruses are well known in the art and are also described herein.Once the host cell has been infected, the helper functions are activatedand rep and cap are produced. Low levels of Rep78 and Rep68 are producedbecause the p5 promoter has been replaced by a minimal promoter or by nopromoter. This, combined with levels of Rep52 and Rep40, both expressedfrom the adenoviral p19 promoter, and the capsid proteins, expressedfrom the AAV p40 promoter, are sufficient to produce high titers ofrAAV.

Methods of producing rAAV using other viral vectors in which Repinterferes with viral replication also may be performed following theteachings of the instant specification.

The rAAV may be purified from the supernatant produced by the host cellsor from cell lysates by any method known in the art or as describedherein. A method of collecting and purifying rAAV is described inExamples 5 and 6.

The method is easily scaled to industrial production because it does notrequire transfection of a large number of host cells to produce rAAV. Ina preferred embodiment, only a single infection of host cells by anadenovirus is required to produce rAAV in large amounts at high titers.See, e.g., Example 5. In another preferred embodiment, host cells areco-infected with two different adenoviruses, one comprising repdownstream of a minimal promoter or no promoter and cap, and the otheradenovirus comprising the rAAV genome. Infection of host cells byadenovirus is highly efficient and may be easily scaled to a largenumber of cells.

The instantly described method produces rAAV at a high titer. In apreferred embodiment, the titer is at least 10² particles per cell;preferably at least 10³ particles per cell; more preferably at least 10⁴particles per cell; and, even more preferably, at least 10⁵ or 10⁶particles per cell. The instant invention also encompasses lysates andsupernatants of host cells comprising rAAV. These lysates andsupernatants differ from those produced by prior art methods because ofthe higher level of rAAV contained therein without concentration.

rAAV Compositions

The rAAV produced by the method of this invention may be formulated as apharmaceutical or pharmacological composition for use for any form oftransient and stable gene transfer in vivo and in vitro. The compositioncomprises at least the rAAV and a pharmaceutically acceptable carrier.The rAAV may be used for in vivo and ex vivo gene therapy, geneticimmunization, in vitro protein production and diagnostic assays.

For gene therapy, the rAAV may be introduced into cells ex vivo or invivo. Where the virus is introduced into a cell ex vivo, the rAAV may beused to infect a cell in vitro, and then the cell may subsequently beintroduced into a mammal (e.g., into the portal vein or into thespleen), if desired. Alternatively, the rAAV may be administered to amammal directly, e.g., intravenously or intraperitoneally. Aslow-release device, such as an implantable pump, may be used tofacilitate delivery of the virus to a cell. Where the virus isadministered to a mammal, the specific cells to be infected may betargeted by controlling the method of delivery. For example,intravascular administration of rAAV to the portal vein or to thehepatic artery may be used to facilitate targeting rAAV to a liver cell.

The rAAV produced by the above-described method may be administered to apatient, preferably suspended in a biologically compatible solution orpharmaceutically acceptable delivery vehicle. A suitable vehicleincludes sterile saline. Other aqueous and non-aqueous sterilesuspensions known to be pharmaceutically acceptable carrier and wellknown to those of skill in the art may be employed for this purpose.

The rAAV is administered in sufficient amounts to infect the desiredcells and provide sufficient levels of transduction and expression ofthe selected transgene (or viral gene products in the case of a vaccine)to provide some level of a corrective effect without undue adverse orwith medically acceptable physiological effects, which can be determinedby those skilled in the medical arts. Conventional and pharmaceuticallyacceptable routes of administration include direct administration to thetarget organ, tissue or site; intranasal; intravenous; intramuscular;subcutaneous; intradermal; oral and other parenteral routes ofadministration. Routes of administration may be combined, if desired.

Dosages of rAAV will depend primarily on factors such as the conditionbeing treated and the selected gene. The dosage may also vary dependingupon the age, weight and health of the patient. For example, aneffective human dosage of rAAV is generally in the range of from about0.5 ml to 50 ml of saline solution containing rAAV at concentrations of1×10⁷ or 1×10⁸ or 1×10⁹ or 1×10¹⁰ or 1×10¹¹ or 1×10¹² or 1×10¹³ or1×10¹⁴ or 1×10¹⁵ or 1×10¹⁶ particles per dose administered. The dosagewill be adjusted to balance the corrective benefits against any adverseside effects. The levels of expression of the selected gene may bemonitored to determine the type and frequency of dosage administration.

The following examples of the present inventions are illustrative only,and are not intended to limit the scope of the invention.

EXAMPLE 1 Cell Lines and Viruses and Maintenance and Propagation Thereof

All cell lines are maintained in Dulbecco's Modified Eagle's Medium(DMEM; Gibco BRL) supplemented with 10% FBS (Hyclone) and 50 μg/ml ofpenicillin, 50 μg/ml of streptomycin, and 10 μg/ml of neomycin (GibcoBRL). Human embryonic kidney cell line 293 is obtained from ATCC(CRL1573). 293-derived 84-31 cells (1) which express adenovirus E1/E4orf6proteins, and HeLa-derived B50 cells (7) which express AAV-2 Rep and Capproteins from the native p5 promoter, are obtained from Dr. GuangpingGao, Institute for Human Gene Therapy, University of Pennsylvania.293-CG3 is a 293-derived cell line carrying stably integrated copies ofAAV ITRs flanking GFP as marker gene (Chen et al., unpublished data).Human adenovirus type 5 (ATCC VR-5) and derived recombinant adenovirusesare propagated on 293 cells and purified through CsCl gradientcentrifugation according to the method of Jones and Shenk withmodification (2).

EXAMPLE 2 Construction of Plasmids and Generation of RecombinantAdenoviruses

Standard recombinant DNA techniques are employed to create recombinantplasmids (3). DNA containing the rep and cap sequences of pAV2 (ATCC37216) between DraIII site (nucleotide 241, upstream of the AAV-2 p5promoter) and NcoI site (nucleotide 4489, downstream of the polyAsignal) is removed and replaced through multiple cloning steps with aDNA cassette containing GFP under the transcriptional control ofelongation factor 1 alpha (EF1α) promoter and upstream of the SV40 polyAsignal to create pAV2cisEFGFP (FIG. 1). The AAV-2 rep and cap geneslocated between a Dra III site (nucleotide 241, upstream of the p5promoter) and a BsaI site (nucleotide 4464, downstream of the polyAsignal) are further subcloned to obtain pAd-p5-RC (FIG. 1). A small DNAfragment between nucleotides 241 and 287 of pAd-p5-RC containing the p5promoter is removed and replaced with a Drosophila melanogaster minimalheat shock protein (HSP) promoter from pIND (Invitrogen) to createpAd-HSP-RC (FIG. 1). Recombinant adenoviruses Ad-p5-RC and Ad-HSP-RC(FIG. 2) are generated according to standard protocols known in the art(see, e.g., Refs. 4 and 12). The recombinant adenoviruses are passagedfive to six times on appropriate mammalian cells to generate a stock ofrecombinant adenovirus that is used for production of rAAV.

EXAMPLE 3 Transfection of 293 Cells and Selection of the 293-CG3 StableCell Line

293 cells are grown to ˜70% confluency in 6-cm tissue culture dishes andco-transfected overnight with 1 μg pIRESIneo and 10 μg pAV2cisEFGFP bythe calcium phosphate transfection method. The monolayer is replenishedwith fresh medium containing 10% FBS and cultured for 24 hours.Following trypsinization, cells are seeded at a 1:20 dilution in freshmedium containing 10% FBS. After incubation for another 24 hours, freshmedium containing 1,250 μg/ml of G418 (Gibco BRL) is added to the cellmonolayer for genetic selection of G418-resistant cells. The mediumcontaining G418 is replaced every 3–4 days to allow formation ofG418-resistant cell colonies. A total of fifty colonies are picked, sixof which demonstrate constitutive GFP expression. These six clones areexpanded and tested for their ability to rescue functional rAAV bytransfection with pBV-EiOV-RC, a plasmid that carries adenovirus E2A,E4ORF6, and VAI genes as well as AAV rep-cap genes. One cell clone,293-CG3, shows high efficiency of rAAV rescue and is expanded and usedfor further experiments.

EXAMPLE 4

PCR Analysis of AAV-2 Rep and Cap Genes Inserted into the AdenovirusGenome

In order to determine the integrity of AAV-2 rep and cap genesrecombined into the adenovirus genome, a polymerase chain reaction (PCR)assay is employed. Four sets of primer pairs overlapping the entire DNAsequence encoding AAV-2 rep and cap genes, as well as the junctionsbetween adenoviral and rep-cap sequences, are synthesized. These primersare:

HC#30 (5′-CGTAACCGAGTAAGATTTGG-3′; SEQ ID NO: 1),

HC#31 (5′-ATGTTGGTGTTGGAGGTGAC-3′; SEQ ID NO: 2),

HC#32 (5′-TGGACCAGAAATGCAAGTCC-3′; SEQ ID NO: 3),

HC#33 (5′-AGCCTTGACTGCGTGGTGGT-3′; SEQ ID NO: 4),

HC#34 (5′-GTACCTGTATTACTTGAGCA-3′; SEQ ID NO: 5),

HC#35 (5′-ACGAGTCAG GTATCTGGTGC-3′; SEQ ID NO: 6),

HC#36 (5′-GGACTTTACTGTGGACACTA-3′; SEQ ID NO: 7), and

HC#37 (5′-GACCCAGACTACGCTGACGA-3′; SEQ ID NO: 8).

To obtain adenoviral DNA for the PCR assay, a miniprep method isemployed. Briefly, 293 cells are grown in 10-cm dishes until nearlyconfluent and then infected with either Ad-HSP-RC or Ad-p5-RC for 3days. Infected cells are harvested, pelleted and lysed in DOC lysisbuffer (100 mM Tris-HCl, pH9.0, 20% ethanol, 0.4% sodium deoxycholate).Cellular nucleic acids are precipitated by spermine-HCl and supernatantis collected. The supernatant is then treated with RNase A and pronase,followed by extraction with phenol-chloroform. Adenoviral DNA in thesupernatant is precipitated with isopropanol and dissolved in TE/RNasebuffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 20 μg/ml RNase).

The PCR assay is performed using the Robocycler Gradient 96 thermalcycler (Stratagene), PCR products are separated on a 1% agarose gel andDNA is stained with ethidium bromide. As shown in FIG. 3, all fourexpected DNA PCR products are obtained when using Ad-HSP-RC DNA astemplate, indicating that the full-length AAV-2 rep-cap DNA is presentin the genome of this recombinant. However, the 880 bp PCR product IIIis not amplified from Ad-p5-RC DNA, suggesting a rearrangement ordeletion event in rep-cap DNA sequences of this recombinant. Theseresults indicate the integrity and stability of HSP-rep-cap DNA afterinsertion into the E1 locus of the adenoviral genome, and point out thatp5-rep-cap DNA sequences inserted into the same locus are unstable.

EXAMPLE 5 Production of rAAV Through Infection of 293-CG3 Cells withAd-HSP-RC

Since Ad-HSP-RC is shown to contain full length AAV-2 rep-cap DNAsequences, its utility to produce rAAV is analyzed. 293-CG3 cells areseeded in 6-well plates at a density of 1.0×10⁶ cells/well. Twelve tofifteen hours later, the culture media is removed from the cellmonolayer and dilutions of Ad-HSP-RC virus in 0.5 ml of serum-free DMEMare added to the cells. Following a 30 min. incubation, an additional2.5 ml of DMEM containing 10% FBS is added and the infection is allowedto proceed for a total of 3 days. Infected cells are harvested andpelleted. Cell pellets are lysed in 1 ml of lysis buffer (50 mMTris-HCl, pH7.4, 1.0 mM MgCl₂, 0.5% DOC) with sonication for 3×1 min.Cell debris is removed by centrifugation at 3,000 rpm using a BeckmanGS-6R centrifuge for 10 min. The lysate supernatant is collected fortitration of rAAV.

To titrate the rAAV in the lysate, 84-31 cells (1) are plated 3–4 hoursbefore use on 24-well plates at a density of 2×10⁵ cells/well. The rAAVlysate is diluted in DMEM containing 10% FBS and heated at 56° C. for 60min to inactivate contaminating Ad-HSP-RC. The rAAV lysate is added tothe monolayer, cells are incubated for approximately 24 hours, andGFP-expressing cells are scored as transducing units (TU).

As shown in FIG. 4, the results demonstrate that rAAV is successfullyproduced through infection of 293-CG3 cells with the recombinantAd-HSP-RC. The rAAV titer increases by increasing the multiplicity ofinfection (MOI) of Ad-HSP-RC. Using an MOI of 250 particles/cell, asmuch as 70 TU/cell of rAAV is produced. However, further increasing theMOI of the Ad-HSP-RC virus does not increase the yield of rAAV. Instead,a slight decrease in rAAV yield is observed, probably due to theincreased cytotoxicity associated with higher MOI of Ad-HSP-RC virus.

EXAMPLE 6 Production of rAAV Through Co-Infection of 293 Cells withAd-HSP-RC and Ad-AAV-LacZ

In the previous example, AAV vector sequences are stably integrated intothe host cell chromosome while rep-cap functions necessary for theirrescue and packaging into rAAV are provided by Ad-HSP-RC. As a separatemeasure to determine the functionality of the Ad-HSP-RC recombinant, AAVvector sequences are delivered exogenously into 293 cells to determinewhether they could be rescued and packaged into rAAV particles followinginfection with Ad-HSP-RC. To perform this experiment, 293 cells areseeded on 6-well plates at a density of 1×10⁶ cells/well. 12–15 hourslater, the cells are co-infected with Ad-HSP-RC and Ad-AAV-LacZ, anE1-deleted adenovirus containing the E. coli lacZ gene flanked by AAV-2ITR's inserted into the E1 locus (4).

To further investigate the effects of varying MOI's of either virus onrAAV yield, the following experiment is performed. Different quantitiesof the two viruses (50 to 450 particles/cell) are mixed together,keeping the total inoculum at a constant 500 particles/cell (FIG. 5).The virus mixture is diluted in 0.5 ml of serum-free DMEM at variousparticle/cell ratios (50:450, 100:400, 200:300, 300:200, 400:100, and450:50) and is added to the cell monolayer. Thirty minutes later, anadditional 2.5 ml of DMEM containing 10% FBS is added and the cells areincubated in the presence of virus inoculum for 3 additional days.

Infected cells are harvested, lysed, and rAAV titrated as described inExample 5 except that rAAV transduction is scored cells by X-galstaining according to standard protocols (5). Cell stain is possiblebecause the rAAV in this experiment carries lacZ as a transgene.Briefly, the rAAV transduced cells are first fixed with 0.5 ml of 0.05%glutaraldehyde for 10 min and then rinsed with 3×0.5 ml of PBS. Fixedcells are stained with 0.5 ml of X-gal solution at room temperatureovernight. The X-gal solution is removed, 0.5 ml of 70% ethanol is addedto terminate the reaction, and blue-staining cells are scored astransducing units.

As shown in FIG. 5, the results clearly demonstrate that co-infection of293 cells by the two recombinant adenoviruses, Ad-HSP-RC andAd-AAV-LacZ, can produce high titers of rAAV in 293 cells. The dataindicate that with decreasing MOI of Ad-HSP-RC and increasing MOI ofAd-AAV-LacZ, the yield of rAAV produced remains relatively steady untilthe Ad-HSP-RC MOI reaches 100 particles/cell or less. Further decreaseof the MOI of Ad-HSP-RC dramatically decreases the rAAV yield,presumably due low levels of Rep and Cap proteins that are produced atthe lower MOI's. On the other hand, increasing the MOI of Ad-AAV-LacZdoes not increase the rAAV yield. While the conventional method for rAAVproduction using plasmid co-transfection is limited in its yield ofrAAV, the current invention provides the means to easily and efficientlyproduce high yields of rAAV.

EXAMPLE 7 Time-Course and Particle Ratio Studies of rAAV ProductionThrough Co-Infection of 293 Cells with Ad-HSP-RC and Ad-AAV-LacZ

To further study the conditions required for optimal production of rAAVthrough co-infection of 293 cells with Ad-HSP-RC and Ad-AAV-LacZ,separate time-course and MOI studies are performed. 293 cells are seededin 6-well plates as described in Example 6 and are infected with bothviruses for different time intervals or at different particles/cellratios. Infected cells are harvested and lysed, and rAAV is titrated asdescribed in Example 6. For the time-course study, 100 particles/celleach of Ad-HSP-RC and Ad-AAV-LacZ are used to infect 293 cells forvarious times.

As shown in FIG. 6, the results demonstrate that rAAV is detected asearly as 24 hours post-infection by the two viruses, but its levelspeaks at 72 hours after infection. More than 200 TU/cell of rAAV isproduced between 48 and 96 hours after infection by the two viruses. Todetermine the optimal MOI of the two viruses required for high levelrAAV production, 293 cells are infected by the two viruses at equalMOI's for 72 hours and harvested and rAAV titers are determined. Asshown in FIG. 7, it is apparent that with increasing input of Ad-HSP-RCand Ad-AAV-LacZ viruses, the yield of rAAV increases up to an MOI of 125particles/cell of each virus. Further increasing the input adenovirusMOI does not increase the rAAV yield and instead results in a slightdecrease of rAAV yield. This may be due to higher cytopathic effectsresulting from higher MOI's of input adenovirus which in turn may affectrAAV yields from the infected cell.

EXAMPLE 8 Analysis of rAAV DNA Replication Following Co-Infection of 293Cells with Ad-HSP-RC and Ad-AAV-LacZ

To analyze the excision and replication of rAAV DNA from the Ad-AAV-LacZhybrid genome, extrachromosomal DNA is analyzed in 293 cells co-infectedby Ad-HSP-RC and Ad-AAV-LacZ using the method of Hirt (6). As a positivecontrol for rAAV DNA rescue and replication, a separate systempreviously shown to produce rAAV at high titers is similarly assayed inparallel. The control system is based on the use of the B50 cell line,that was previously created by stably transfecting into HeLa cells arep/cap-containing plasmid utilizing endogenous AAV-2 promoters (7).rAAV production in this cell line occurs in a two-step process: B50cells are initially infected with sub100r, an adenovirustemperature-sensitive mutant in the E2b gene, to induce rep and capexpression and provide helper functions. 24 h later, cell are infectedby a hybrid E1-deleted adenovirus in which the AAV vector sequence iscloned in the E1 region of a replication-defective adenovirus. In thepresence of Rep and Cap proteins expressed by the cell, as well asadenoviral helper functions expressed from sub 100r, the rAAV genomedelivered by the hybrid vector is rescued, replicated and encapsidatedinto rAAV particles (7).

293 cells are grown in 10-cm dishes to subconfluency and are eitherco-infected with Ad-HSP-RC plus Ad-AAV-LacZ or with Ad-p5-RC plusAd-AAV-LacZ at 200 particles/cell of each virus. Positive controlB50/sub100r experiments are carried out in a similar fashion except thatAd-AAV-LacZ is added to B50 cells 24 hours after addition of sub 100r,each at 1,000 particles/cell. Negative controls include single virusinfections of 293 cells with either Ad-p5-RC, Ad-HSP-RC or Ad-AAV-LacZalone. Infected cells are harvested 72 hours post-infection and lysed in0.85 ml of Hirt solution (10 mM Tris-HCl, pH 7.4, 100 mM EDTA, 0.6%SDS). The lysate is mixed with 0.25 ml of 5 M NaCl and incubated at 4°C. overnight. After centrifugation at 14,000 RPM for 40 min. in aSorvall centrifuge, supernatant is collected and extracted three timeswith phenol-chloroform. The low molecular weight DNA is precipitated byisopropanol, dissolved in TE/RNase buffer, fractionated byelectrophoresis on a 0.8% agarose gel and stained with ethidium bromide.

The results of this experiment are presented in FIG. 8. Co-infection of293 cells by Ad-HSP-RC and Ad-AAV-LacZ results in the generation of DNAbands of 4.8 and 9.6 kb which correspond to monomeric and dimeric formsof replicating, double-stranded rAAV DNA (FIG. 8, lane 4). The positivecontrol also shows the same two DNA species following infection of B-50cells with sub100r and Ad-AAV-LacZ (FIG. 8, lane 6). Neither DNA band isobserved in negative controls in which 293 cells are infected singlywith either Ad-p5-RC (FIG. 8, lane 1), Ad-HSP-RC (FIG. 8, lane 2), orAd-AAV-LacZ (FIG. 8, lane 3). Furthermore, co-infection of 293 cells byAd-p5-RC and Ad-AAV-LacZ also does not result in the formation of eitherrAAV DNA species (FIG. 8, lane 5), suggesting that Ad-p5-RC is defectivein its rep/cap functions for rescue or replication of rAAV DNA.

To confirm that the 4.8 and 9.6 kb extrachromosomal DNA species detectedin FIG. 8 does indeed contain the lacZ transgene, and to confirm thatlow quantities of such DNA species are indeed absent in negative controllanes, a Southern blot analysis is performed. Hirt-extracted DNA,isolated from either 293 cells co-infected by Ad-HSP-RC and Ad-AAV-LacZor B50 cells infected by sub 100r and Ad-AAV-LacZ, is separated on a0.8% agarose gel (FIG. 9A). The gel is transferred to a nitrocellulosemembrane and hybridized with a digoxigenin labeled, lacZ DNA probe usingthe DIG High Prime DNA Labeling Detection Starter Kit II from BoehringerMannheim (FIG. 9B). A lacZ DNA fragment, isolated from a bacterialplasmid, is used as positive control for the Southern blot (FIGS. 9A and9B, lane 1).

Both monomeric and dimeric forms of replicating rAAV DNA, isolated fromeither 293 cells co-infected by Ad-HSP-RC and Ad-AAV-LacZ (FIGS. 9A and9B, lane 5) or B50 cells infected by sub100r and Ad-AAV-LacZ (FIG. 9Aand 9B, lane 7), hybridize to the lacZ probe. Southern blotting does notdetect any replicated rAAV DNA species following co-infection of 293cells by Ad-p5-RC and Ad-AAV-LacZ (FIGS. 9A and 9B, lane 6), or from thenegative controls which include Hirt DNA from 293 cells infected singlyby either Ad-HSP-RC (FIGS. 9A and 9B, lane 2), Ad-p5-RC (FIGS. 9A and9B, lane 3), or Ad-AAV LacZ (FIGS. 9A and 9B, lane 4). Taken together,these results indicate that co-infection of 293 cells by Ad-HSP-RC andAd-AAV-LacZ results in rescue and replication of rAAV DNA from theAd-AAV-LacZ genome, and that replicating rAAV DNA indeed contains thelacZ transgene. Moreover, the lack of replicating rAAV DNA afterco-infection of 293 cells by Ad-p5-RC and Ad-AAV-LacZ supports earlierobservations that Ad-p5-RC is indeed defective in its rep/cap functionsfor rescue or replication of rAAV DNA.

EXAMPLE 9

-   -   Analysis of Rep/Cap Protein Expression in 293 Cells Infected        with Ad-HSP-RC Virus

To analyze the expression of Rep and Cap proteins in cells infected byAd-HSP-RC virus, Western blotting is performed. 293 cells are grown in10-cm plates to subconfluency and are either infected with Ad-HSP-RC,Ad-AAV-LacZ, or Ad-p5-RC using 200 particles/cell of each virus. 72hours post-infection, cells are harvested, pelleted, and processed asdescribed by Xiao et al. (8). Briefly, the infected cell pellet from one10-cm dish is lysed in 0.5 ml lysis buffer containing 10 mM Tris-HCl, pH8.2, 1% Triton X-100, 1% SDS and 150 mM NaCl by sonication. Samples areseparated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 4%˜20%gradient polyacrylamide gel and transferred to a nitrocellulosemembrane. The Rep proteins are detected using monoclonal antibody 303.9and Cap proteins are detected using monoclonal antibody B1 (AmericanResearch Products, Inc.), both at a 1:20 dilution. Protein-antibodycomplexes are visualized using the ECL™ Western Blotting Analysis System(Amersham).

As shown in FIG. 10, Rep and Cap proteins are expressed in 293 cellsinfected with Ad-HSP-RC alone (Lane 3), or co-infected with Ad-HSP-RCand Ad-AAV-LacZ (Lane 5), but not in 293 cells infected with Ad-p5-RCalone (Lane 1), Ad-p5-RC plus Ad-AAV-LacZ (Lane 2), or Ad-AAV-LacZ (Lane4). Levels of Rep52 and Rep40 proteins expressed following Ad-HSP-RCinfection are much higher than those of Rep78 and Rep68, a phenomenonthat has been previously reported to contribute to higher levels of rAAVproduced (9). The low basal transcriptional activity of the HSP promotermay indeed play an important role in the success of creating theAd-HSP-RC recombinant adenovirus since it is known that expression ofAAV Rep proteins may interfere with adenovirus replication (10). Lowlevel expression of these proteins may minimize the interference butprovide enough Rep function for excision and replication of the rAAVgenome.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

REFERENCES

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1. An adenovirus vector for the manufacture of rAAV, wherein theadenovirus vector comprises an AAV rep gene, and wherein the AAV p5promoter is deleted upstream of the AAV rep gene and said vectorcontains a minimal promoter in place of the p5 promoter, wherein theminimal promoter contains a TATA box as its only regulatory element. 2.An adenovirus vector for the manufacture of rAAV, wherein the adenovirusvector comprises an AAV rep gene, and wherein the AAV p5 promoter isdeleted upstream of the AAV rep gene and said vector contains a minimalDrosophila heat shock promoter in place of the p5 promoter.
 3. Anadenovirus vector for the manufacture of rAAV, wherein the adenovirusvector comprises an AAV rep gene, and wherein the AAV p5 promoter isdeleted upstream of the AAV rep gene and said vector contains a minimaladenoviral E1b promoter in place of the p5 promoter.
 4. The adenovirusvector of claim 1, 2, or 3, wherein the adenovirus vector furthercomprises an AAV cap gene.
 5. The adenovirus vector of claim 4, whereinthe rep gene and the cap gene are inserted in place of at least aportion of one or more of the E1, E3 or E4 genes of adenovirus in alocus of the adenovirus vector.
 6. The adenovirus vector of claim 5,wherein both the rep gene and the cap gene are inserted within the samelocus of the adenovirus vector.
 7. The adenovirus vector of claim 5,wherein the rep gene and the cap gene are inserted within different lociof the adenovirus vector.
 8. The adenovirus vector of claim 4, whereinthe rep and cap genes are from different AAV serotypes.
 9. Theadenovirus vector of claim 4, wherein the rep and cap genes are from thesame AAV serotype.
 10. A method for producing rAAV, comprising the stepsof: a) infecting a host cell comprising a rAAV genome with theadenovirus according to claim 1, 2 or 3 wherein the infected host cellcomprises helper functions and Cap coding sequences; b) growing the hostcells under conditions in which rAAV is produced; and c) optionallycollecting the rAAV from the host cells.
 11. The method according toclaim 10, wherein the rAAV genome is stably integrated in a chromosomeof the host cells.
 12. The method according to claim 10, wherein thehost cell comprises an adenovirus vector comprising the rAAV genome andsaid host cell is co-infected with the adenovirus vector comprising therep gene.
 13. The method according to claim 10, wherein the adenovirusvector provides a helper function for rAAV production.
 14. The methodaccording to claim 13, wherein said helper function is provided by atleast one gene product selected from the group consisting of adenoviralgenes E1A, E1B, E2A, E4orf6 and VAI, or at least one gene productselected from the group consisting of HSV type 1 genes UL5, UL8, UL52,and UL29.
 15. The method according to claim 10, wherein the host cell isa 293 cell.
 16. The method according to claim 10, further comprising thestep of purifying the rAAV.
 17. The method according to claim 10,wherein the host cells are selected from CHO, BHK, MDCK, 10T1/2, WEHIcells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRC5, A549, HT1080, 293,B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7, HER, HEK, HEL, or HeLa cells.18. A method for producing rAAV, comprising the steps of, growing a hostcell comprising a rAAV genome and an adenovirus according to claim 1, 2or 3 under conditions in which rAAV is produced and wherein the hostcell further comprises helper functions and Cap coding sequences; andoptionally collecting the rAAV from the host cells.
 19. The adenovirusvector of claim 1, 2, or 3 wherein Rep78 and Rep68 are produced at lowerlevels than Rep52 and Rep40 in a cell into which the adenovirus vectorhas been introduced.